Causes and elimination of erratic blanks in enzymatic metabolite assays involving the use of NAD+ in alkaline hydrazine buffers: Improved conditions for the assay of l-glutamate, l-lactate, and other metabolites
✍ Scribed by Paul C. Engel; Janet B. Jones
- Book ID
- 102985598
- Publisher
- Elsevier Science
- Year
- 1978
- Tongue
- English
- Weight
- 630 KB
- Volume
- 88
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
In the alkaline hydrazine buffers frequently used for enzymatic metabolite assays, NAD+ undergoes reactions which give rise to products that absorb light at 340 nm. These "blank" reactions hinder accurate metabolite measurements. At least two reactions are involved, a very rapid one which results in a new absorbance band maximal at 316 nm and a slow one in which an absorbance band maximal at 342 nm develops over many hours. The first of these processes is strongly pH dependent and is responsible for the high initial absorbance of metabolite assay reaction mixtures. The second reaction, which is responsible for drifting endpoints in metabolite assays, appears to be potentiated by heavy metal ions and suppressed by EDTA. It is suggested that assays involving the use of NAD+ in hydrazine buffers should be carried out in the presence of high concentrations of EDTA and that the pH should not be higher than is absolutely necessary to ensure a quantitative assay. The standard assays for L-lactate and L-glutamate have been substantially improved by adopting these measures.